Introduction
Transformation of yeast with yeast artificial chromosomes (YACs) has traditionally
been performed by a PEG-spheroplast procedure.1,2 However, the procedure
is complicated, often unreliable, and transformation efficiencies are
relatively low when compared to more recently developed transformation
methods. An additional concern is that the smaller YACs of any given ligation
mixture are cloned preferentially, therefore prior size fractionation
with sucrose gradients to enrich for larger molecules, or treatment with
spermidine to improve transformation efficiency of larger molecules, is
required to obtain reasonable efficiencies with larger YACs. Several electroporation
protocols have recently been reported for the transformation of Saccharomyces
cerevisiae with supercoiled plasmids.3-6 In an attempt to improve the
currently used YAC cloning procedure, we investigated the electroporation
of S. cerevisiae strain AB1380 (Mat a, ade2-1, can1-100, lys2-1, trp1,
ura3, his5, psi + ) with a 14.4 kb mini-YAC. Initial experiments showed
transformation efficiencies two-fold better than those reported in a previously
published YAC electroporation protocol.7

Materials and Methods
The YAC used in our experiments was constructed by the insertion of a
4.6 kb lambda MluI fragment into the MluI-SUP4 cloning
site of pYAC-RC. 8 E. coli was transformed by the resulting
16.1 kb plasmid, the DNA was isolated, purified over cesium chloride,
and restriction d
igested with BamHI, thereby freeing the telomeres
and resulting in a 14.4 kb linear DNA molecule. The restriction-digested
DNA was microdialized (Millipore VS, 0.025 m) against TE (10 mM Tris-HCl,
1 mM EDTA, pH 7.5) for 20 minutes, ethanol precipitated, and suspended
in TE. The electroporation protocol used for our experiments was an adaptation
of the one devised by Becker and Guarente.5 Exponentially growing
cells were harvested by centrifugation, washed twice in water (4 C),
once in 1 M sorbitol (4 C), and finally resuspended in a 2/3 pellet volume
of 1 M sorbitol (4 C). A 40 l aliquot of cells was gently mixed with
1-5 l of DNA (0.1 g) just before electroporation. A Gene Pulser apparatus
connected to the Pulse Controller accessory was used to pulse the sample
in a chilled 0.2 cm cuvette, generating a pulse with a field strength
of 7.5 kV/cm (1.5 kV), and a time constant (τ) of approximately 4.2 msec
(25 F and 200 Ω). Ice cold 1 M sorbitol (1 ml) was added immediately
after pulse delivery, and 150 l aliquots were plated on SD ura- plates.
Transformants were visible after 3 days of incubation at 30 C.

Results and Discussion
Transformations with the small YAC have yielded approximately 700 transformants
/ g of DNA. Transformations with a supercoiled 5 kb plasmid yielded up
to 1.6 x 104 transformants / g of DNA. A number of parameters were investigated
in attempt to increase efficiencies. We found that 0.1 g of DNA in 1-5
l of TE or water was the optimum amount for 40 l of cells (5 x 108 cells).
More DNA had an adverse effect on transformation efficiency and less DNA
did not seem to saturate the reaction (Figure 3). Spermidine
-treated DNA
did not yield any transformants. Exponentially growing cells gave the
most consistent and highest transformation efficiencies.

A possible problem associated with the high voltage electroporation of
very large DNA fragments such as YACs is that the molecules are most likely
sheared before they have a chance to enter the cell. Lower voltage experiments
with the Gene Pulser apparatus (which generates an exponential decay pulse)
have a less disruptive effect on large DNA molecules, but they also yield
lower transformation efficiencies.3,4 The use of alternative waveforms
may improve on current results.6 The S. cerevisiae strain used here (AB1380)
is traditionally employed in PEG-spheroplast transformations and we do
not know whether this strain is also a good electroporation candidate.
Other strains that have proven to yield high transformation rates might
further improve the results presented here.

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